CS216997B1 - Method of isolating silymarin complex - Google Patents

Abstract

Method for isolating the silymarin complex from the fruits of milk thistle (Silybum marianum /L./ Gaertn.) by extraction with aqueous ethanol at elevated temperature or with aqueous acetone at normal to slightly elevated temperature, concentration of the extract and transfer of the substances to 60% vol. aqueous ethanol, defatting the solution with a non-polar organic solvent, such as petroleum ether or gasoline, and subsequent extraction of silymarin with chloroform. The silymarin complex is separated from the concentrated chloroform extract by dilution with water.

Description

The invention relates to a process for the isolation of silymarin, a complex of flavonoids, so-called flavolignans, derived from flavanonol taxifoline and contained in the fruit (samara) of Silybum marianum (L. / Gaertn.). This complex is used as a medicine for various liver diseases. It is characterized by a stimulating effect on liver cell metabolism and a protective action on their membranes (G. Vogel et al., Arzneimittel-Forsch. 25, 82, 1975). In particular, the isomeric compounds silybin, silydianin and silychristin form an essential component of the complex.

Extraction of silymarin from milk thistle fruits is extremely difficult due to three factors: the first is a high oil content in the drug, up to 30% by weight, the second is the solid binding of silymarin in the cell and the resulting slow diffusion into the extracting agent; silymarin is contained exclusively in the surface parts of the achenes, while all the oil, together with the starch grains, is contained in the endosperm. The third factor is the slight selection of purification methods. The solubility of silymarin is limited to a few polar solvents, the amorphous nature of the complex only allows precipitation purging procedures; acid-base interventions cannot be used for the sensitivity of flavolignans.

In the theoretical work dealing with the chemical nature of silymarin and later its components, it was not necessary to take into account the laboriousness and lengthyness of the isolation process. As a rule, finely ground plant material was first carefully defatted with a non-polar organic solvent and then extracted for a long time with ethyl acetate or acetone (B. Janiak, R. Hansel, Planta Medica 8, 71, 1960; H. Wagner et al., Naturwissenschaften 52, 305, 1965). ).

For industrial purposes, however, it was necessary to eliminate or at least technologically pre-defat the drug and speed up the extraction of silymarin. According to these criteria it is possible to divide the existing processes, mostly patent protected. In some, defatting is omitted and the ground material is extracted with acetone (French Pat. No. 2,181,188), ethyl acetate-methanol-water (Belgian Pat. No. 766,228), by combining polyethylene glycol 200 with ethanol or acetone ( German Pat. No. 2,017,789) or subsequently by several solvents of the eluotropic series (Japanese Pat. No. 8 09,211). The major part of the extracted oil is generally separated after diluting the concentrated leach with water. Furthermore, defatting of finely ground drug with dichloromethane, subsequent extraction with acetone and refining of the silymarin pool on powdered polyamide have been described (GDR Pat. No. 112,261).

According to another method, the whole fruit is freed of most of the oil by pressing, which also disrupts the cells and facilitates subsequent extraction. The crushed material is then extracted with ethyl acetate. The concentrate obtained by evaporation is freed from oil residues and also enriched by countercurrent distribution in a petroleum ether-methanol-water system on a series of flow separators (German Pat. Nos. 1,767,666 and 1,923,082; U.S. Pat. No. 162,722). A content of 70 to 80% by weight is declared in the final silymarin substance after this isolation procedure. silymarin type flavonoids.

By analyzing and accurately reproducing the prior art methods of preparing silymarin, it has been found that silymarin can be satisfactorily extracted from a non-defatted drug with either a ketone solvent (acetone) or a mixture of organic solvents with a wide range of dielectric constants. In no case has the beneficial effect of fats on the extraction of silymarin mentioned in French Pat. No. 2 18, 188.

The disadvantages of the known processes are either the relatively high cost or poor availability of the solvent (polyethylene glycol) and the difficult recovery of the extraction medium in the case of solvent combinations. In another case, the pre-pressing of most of the oil is advantageous, but the subsequent extraction with ethyl acetate is considerably non-selective and, moreover, the ethyl acetate decomposes during extraction and concentration of the extract. The complex resultant ethyl acetate-acetic acid-ethanol system then extracts a number of difficult ballasts from the drug. Also, solvent consumption and the cost of the countercurrent enrichment of the silymarin concentrate are not proportional to the refining result.

The issue of drug extraction and enrichment of the silymarin summary is partially addressed by a process (Polish Pat. Nos. 89,368 and 97,285), which makes use of the possibility to distinguish part of the oil from moistened and swollen achenes or to treat separately peeled surface parts of spawned and endosperm. By this procedure, silymarin is extracted from partially defatted material or fruit surface with 90% aqueous methanol, the concentrated extract is defatted with petrol, precipitated with water, and the dried intermediate is enriched by extraction with dichloromethane or acetone. The disadvantage of this process is the relatively complex treatment of the drug prior to the actual extraction of the silymarin, for example by squeezing swollen achenes, hot pressing, grinding, sieving and finally technologically uncertain enrichment of the silymarin concentrate by extraction of the solid with an organic solvent.

The above-mentioned disadvantages of the prior art have been overcome by the development of a process for the isolation of silymarin from the milk thistle fruit (Silybum marianum (L../Gaertn.)) According to the invention, which consists in extracting whole crushed fruits or their separated surface parts with aqueous ethanol at a concentration. 80 to 90% v / v at a temperature of 50 ° C to the boiling point of the extracting agent or aqueous acetone at 90 to 98% v / v at a temperature of 15 to 35 ° C, the extract after conversion into an oily residue is converted into a silymarin complex solution in 50 to 60% aqueous ethanol, the solution is freed from oil by extraction with a non-polar organic solvent, preferably petroleum ether, followed by extraction of the aqueous-ethanolic phase with an organic solvent of the chlorinated hydrocarbon group, preferably chloroform, concentrated to 10-20% of its original volume; of this concentrate, the silymarin k is precipitated by dilution with water omplex.

The extraction of the starting drug is expediently carried out with aqueous ethanol at a concentration of 80 to 90% by volume in a ratio of 1: 8 to 1:12, preferably 1:10 (w / v) or aqueous acetone at a concentration of 90 to 98% by volume in a ratio of 1: : 4 to 1:10, preferably 1: 5 to 1: 7 (w / v). The process according to the invention was based on new knowledge and experience from all production stages. In the initial phase, it is important that the drug does not crush too much; Silymarin is contained exclusively in the surface parts of the fruit. This prevents the release of starch from the internal parts of the fruit, since otherwise released starch greatly slows the solvent flow through the drug. It is then essentially irrelevant to the further procedure and result whether partially defatted or non-defatted drugs, crushed fruits or separate packages are used. It is important to disrupt cells containing silymarin by appropriate means.

For economic reasons, it is desirable to be able to extract the drug with a cheap and available solvent that is easy to recover. The extract must be further processed in a simple, reproducible and technologically pure manner. As experimentally verified, extraction with 80-90% aqueous ethanol at elevated temperature or similarly effective extraction with 90-98% aqueous acetone without heating is most preferred.

When renumbering the extract, it is important to keep the silymarin complex up to the final substance stage under conditions such that a viscous resin mass that is difficult to handle on an industrial scale is always avoided. The actual enrichment is then best carried out on the principle of partition equilibria in the liquid-liquid system. Both of these requirements are met in the process according to the invention. A mixture of a monohydric aqueous alcohol with a chlorinated hydrocarbon, preferably an ethanol-water-chloroform mixture, has proved to be a very advantageous solvent system for enriching the silymarin complex. The enriched silymarin complex passes into the heavy (chloroform) phase of the system, the polar ballast substances and especially the pigments remain in the light phase.

In the preparation of the final substance, the heavy phase can be concentrated to a viscous homogeneous solution which, in a known manner, provides a coarse, well filterable suspension by mixing with an excess of cold water. Drying, preferably under vacuum at a temperature of up to 30 ° C, yields a silymarin complex of suitable physical properties, free of residual organic solvents, containing at least 70% by weight. flavolignans determined by the photometric method according to Wagner with sp. (Arzneimittel-Forsch. 1968, 8, 696). This output stage is very important. Experience has shown that silymarin precipitated in or washed with organic solvents very strongly attracts their residues even under intensive vacuum drying. This eliminates the disadvantage of the process described in cit. Polish pat. in which the summary is enriched in the form of a solid substance by abetone or dichloromethane.

The choice of the drug extraction medium in the process of the invention is to some extent determined by the refining process described in more detail below. The distribution coefficient of silymarin (flavolignans) as well as the refining effect is significantly favorable in the ethanol-water-chloroform system. A number of concentration and temperature dependencies have resulted in optimum extraction conditions, namely aqueous ethanol at a concentration of 80 to 90% by volume and a temperature of from 50 ° C to the boiling point of the extractant, preferably 55 to 60 ° C. For extraction, usually 10 parts by volume of extractant per 1 part by weight of the starting drug can be extracted continuously (counter-current) or batchwise. In this case, it is best to extract twice by 5 parts by volume of solvent, with stirring, for 5 hours each. If hot extraction of the drug is not feasible, aqueous ethanol can be replaced by 90-98% aqueous acetone. Extracts of 5 to 7 parts by volume of extractant per 1 part by weight of the starting drug are usually sufficient. After distilling off the acetone, the concentrate obtained is further enriched by the same procedure in the ethanol-water-chloroform system.

The aqueous-ethanol extract of a non-defatted, coarsely crushed drug is a brown, viscous solution containing about 60% ethanol by volume and a partially precipitated oil after a concentration of 1:10. In order to quantitatively remove the oil, it is sufficient to shake the concentrate several times (for example, three times) with petroleum ether, in an amount of, for example, 0.2 parts by volume, based on the volume of the concentrate. It is advisable to determine the ethanol concentration beforehand and to adjust it to a value of 60% by volume, which is optimal for the subsequent shaking of silymarin. The defatted concentrate is sufficient to shake once 0.2 parts by volume and twice by 0.1 parts by volume of chloroform, the resulting volume of combined shakes being more than twice the volume of the chloroform used. Chloroform can also be replaced by another chlorinated hydrocarbon such as cheap and readily available 1,2-dichloroethane, but its consumption is 2.5 times greater.

The aqueous acetone extract, after distilling off the acetone, yields a concentrate which is dissolved directly in the distillation vessel in a volume of 60% aqueous ethanol corresponding to the weight of the starting drug. The solution obtained is then shaken with chloroform as described above.

The details of the process according to the invention are evident from the following examples, which are illustrative but not limiting.

Example 1

100 kg of coarsely crushed thistle fruit, particle size 0.5-2.0 mm, is extracted with 500 liters of 80% aqueous ethanol with stirring at 55-60 ° C for 5 hours. The extract is separated and the drug is digested in the same manner once more with an equal volume of aqueous ethanol. If a countercurrent hot extraction device is available, the drug may be extracted at the indicated temperature at a rate such that the solvent contact time is 0 to 12 hours. The extract is concentrated under reduced pressure to a volume of 90 to 100 liters (i.e. in a ratio of about 1:10). The distillate is used for further extraction after adjusting the ethanol concentration to 80% by volume. The ethanol content of the concentrate is determined by a suitable method, such as distillation test or gas chromatography, and adjusted to 60% by volume. The dark brown solution containing a partially precipitated oil is extracted three times with 20 liters of light petroleum each. Petroleum ether is recovered from the combined extracts. The oil-free aqueous-ethanol phase is extracted with three 20, 10 and 10-liter chloroform portions, with most of the brown ballasts remaining in the upper phase of the system. The combined lower phases, colored light brownish green, have a volume of about 100 liters. It is concentrated under reduced pressure to 10 liters, i.e. to 1/10 of the weight of the drug (v / v), and the concentrate is discharged to 60 liters of water at a temperature of up to 25 ° C over a period of 5 to 30 minutes. The resulting suspension is suctioned off through a cloth, washed with water and dried, under vacuum at an external temperature of initially to 40 ° C, after evaporation of the major part of water gradually up to 60 ° C. The last drying stage can also be carried out in a hot-air dryer.

The silymarin complex obtained is a beige powder containing at least 70 wt. flavolignans, determined by the method of Wagner with sp. (Arzneimittel-Forsch., Loc. Cit.). Yield 3,650 g.

Example 2

100 kg of peeled surface parts of the milk thistle fruit are extracted twice with 500 liters of 80% aqueous ethanol as in Example 1. In this case, the extract is concentrated to 200 to 300 liters, since it contains more silymarin which could be prematurely precipitated as a resin. A double extraction of 20 liters of light petroleum is sufficient to remove the oil residue. The 60% v / v aqueous-ethanol phase is extracted with three 40, 20 and 20 liter chloroform portions. The bottom phase is concentrated to 20 liters and the concentrate is precipitated with 120 liters of water. Further processing is as in Example 1.

The silymarin complex obtained is of the same quality as in Example 1. The yield is 7000 g.

Example 3

100 kg of coarsely crushed milk thistle fruit with a particle size of 0.5 to 2.0 mm are extracted twice with 350 to 500 liters of 95% aqueous acetone with stirring at 25 ° C for 5 hours each. The combined extracts are concentrated under reduced pressure to an oily residue which is stirred with 90 liters of 60% aqueous ethanol. This solution was shaken with petroleum ether and chloroform and further processed as in Example 1. The yield was 4080 g.

Claims (2)

SUBJECT OF THE INVENTION A process for the isolation of a silymarin complex from the milk thistle fruit (Silybum marianum (L../Gaertn.)), Characterized in that either the whole crushed fruit or its separated surface portions are extracted with 80 to 90% by volume aqueous ethanol at a temperature of from 50 ° C to the boiling point of the extracting agent or aqueous acetone at a concentration of 90 to 98% vol. at 15 to 35 ° C, the extract after processing into an oily residue is converted into a solution of the silymarin complex in 50 to 60% aqueous ethanol, the solution is freed from oil by extraction with a non-polar organic solvent, preferably petroleum ether, followed by extraction with the organic solvent from the group of chlorinated hydrocarbons, preferably chloroform, the extract is concentrated to 10 to 20% of its original volume and from this concentrate the silymarin complex is diluted by dilution with water. 2. A method according to claim 1, wherein the extraction of the starting drug is carried out with aqueous ethanol at a concentration of 80-90% by volume in a ratio of 1: 8 to 1:12, preferably 1:10 (w / v) or aqueous acetone. a concentration of 90 to 98% by volume in a ratio of 1: 4 to 1:10, preferably 1: 5 to 1: 7 (w / v).